Polymerase chain reaction (PCR) is a process for amplifying quantities of double-stranded deoxyribonucleic acid (DNA). Real-time detection of DNA amplification during the PCR process provides quantitative data for amplifiable DNA target sequences by relating the number of temperature cycles during thermal cycling to reach a concentration threshold (Ct) of the target sequence to the amount of target DNA present at the beginning of the PCR process. In real-time detection, the time required to read a sample during annealing can impact the overall run time. Currently, it takes about 2 hours to run PCR in a standard cycling instrument and about 40 minutes in a fast cycling instrument. In an effort to reduce this time, an excess of enzymes can be added to each well thereby decreasing the reaction time. However, the reaction time can only be decreased to an extent, and the excess enzyme increases the cost of the experiment.
Currently, the 60° C. annealing temperature can be used to take fluorescent reads because it is a stable temperature at which to read spectrally distinguishable species, such as fluorescent dyes. There are chemical and physical limitations to the use of spectrally distinguishable species. One of these limitations is the variation of excitation wavelengths of different colored species. As a result, simultaneously using two or more spectrally distinguishable species with different excitation wavelengths requires multiple excitation light sources. Moreover, the dyes can change in intensity as well as in spectrum with temperature. Because it is not possible to read every well and/or every channel simultaneously, a 20 or 30 second hold exists.
It may be desirable to reduce the time between each cycle by calibrating the temperature of each well thereby reducing the hold time.